Preadipocyte proliferation and / or differentiation promoter containing helical lactone derivatives

Abstract

The present invention discovers a compound contained in an arnica extract that promotes the proliferation and / or differentiation of preadipocytes, and provides cosmetics, foods, pharmaceuticals, etc. and / or raw materials for use in them for increasing the volume of the fat layer and creating a beautiful appearance using the compound. The present invention relates to a preadipocyte proliferation and / or differentiation promoter, which contains a compound represented by the following formula (I) as an active ingredient. (In the formula, R is a hydrogen atom or an acyl group having 1 to 5 carbon atoms.)

Description

[0001] Cross Reference

[0002] This application claims priority based on Japanese Patent Application No. 2022-082055 filed in Japan on May 19, 2022, and the contents described in the application are incorporated herein by reference. Technical Field

[0003] The present invention relates to a proliferation and / or differentiation promoter for preadipocytes containing a helenalin derivative. Background Art

[0004] Fat cells are found in the lowest layer of the skin structure consisting of three layers: epidermis, dermis, and subcutaneous tissue, and constitute an energy storage tissue that stores fat in the form of triglycerides. Fat tissue stimulates other tissues by secreting various adiponectins such as adiponectin, which is known as a longevity hormone. Fat tissue not only has its physiological functions, but also has the function of creating beautiful appearance, such as showing a sense of fullness in women and improving wrinkles through local accumulation of fat.

[0005] Although adipocytes have important physiological and aesthetic functions as described above, adipocytes are considered harmful due to the recent trend of weight loss. Therefore, cosmetics, foods, and pharmaceuticals containing ingredients that specifically inhibit the proliferation of preadipocytes and inhibit differentiation into adipocytes have been developed. On the other hand, in order to inhibit the aging phenomenon of the skin, etc., in order to promote the proliferation of myoblasts that serve as the basis of the muscles located inside the skin, a skin external composition containing an arnica extract and a red algae extract is disclosed (for example, refer to Patent Document 1).

[0006] Extracts extracted from Arnica montana flowers of the Asteraceae family have been used as Chinese herbal medicines with anti-inflammatory and blood circulation effects since ancient times. It is also reported that the Arnica montana extract has the effect of increasing fat cells, increasing the volume of the fat layer, and improving the tension and elasticity of the skin (for example, see Patent Document 2). However, the active ingredients contained in the Arnica montana flower extract that are related to the promotion of the proliferation and differentiation of preadipocytes are not yet clear.

[0007] Prior art literature

[0008] Patent Literature

[0009] Patent Document 1: Japanese Patent Application Publication No. 2011-32233

[0010] Patent Document 2: Japanese Patent Application Publication No. 2020-023476 Summary of the invention

[0011] Technical problem to be solved by the invention

[0012] The technical problem to be solved by the present invention is to discover a compound contained in an arnica extract that promotes the proliferation and / or differentiation of precursor adipocytes, and to provide cosmetics, foods, pharmaceuticals, etc. that use the compound to increase the volume of the fat layer to create a beautiful appearance, and / or raw materials for use therein.

[0013] Technical solutions for solving technical problems

[0014] The present invention was made to solve the above-mentioned problems and was made based on the discovery that a specific compound fractionated and purified from an Arnica montana flower extract using column chromatography or the like promotes proliferation and / or differentiation of preadipocytes.

[0015] That is, the present invention includes the following embodiments.

[0016] (1) A preadipocyte proliferation and / or differentiation promoting agent comprising a compound represented by the following formula (I) as an active ingredient.

[0017] [Chemistry 1]

[0018]

[0019] (In the formula, R is a hydrogen atom or an acyl group having 1 to 5 carbon atoms.)

[0020] (2) The agent for promoting proliferation and / or differentiation of preadipocytes according to (1), wherein the compound is 6-O-methacryloylhelene lactone or 6-O-isobutyrylhelene lactone.

[0021] (3) The agent for promoting proliferation and / or differentiation of preadipocytes according to (1), wherein the compound is helianthus annuus lactone or 6-O-acetylhelianthus annuus lactone.

[0022] (4) The agent for promoting proliferation and / or differentiation of preadipocytes according to (1), wherein the compound does not contain 6-O-tigloyl-helidolide, or contains 6-O-tigloyl-helidolide in an amount less than the total amount of the active ingredient in terms of mass ratio.

[0023] (5) A skin external preparation for promoting proliferation and / or differentiation of preadipocytes, comprising the compound represented by the above formula (I) as an active ingredient.

[0024] (6) The skin external preparation according to (5), wherein the content of each active ingredient is at least 0.1×10 -9 quality%.

[0025] (7) The skin external preparation according to (5) or (4), which does not contain 6-O-tiglylhexadecene lactone, or contains about 1×10 -76-O-tiglyloylhexanolide is contained at a concentration of mass % or less.

[0026] Effects of the Invention

[0027] The agent for promoting proliferation and / or differentiation of preadipocytes of the present invention can be provided as cosmetics, foods, pharmaceuticals, etc., and / or raw materials to be blended therein, for promoting proliferation and differentiation of preadipocytes and increasing the volume of the fat layer to create a beautiful appearance. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] Figure 1 A scheme for isolating and purifying active ingredients from an Arnica montana extract is shown.

[0029] Figure 2 The results of proliferation test and differentiation test of human subcutaneous preadipocytes of each fraction obtained by diethyl ether extraction and silica gel open column chromatography separation of Arnica montana extract are shown.

[0030] Figure 3 The results of proliferation and differentiation tests on human subcutaneous preadipocytes of Compound 1 and Compounds 3 to 10 separated from the DEFr.4-2 fraction using silica gel open column chromatography, preparative HPLC and the like are shown.

[0031] Figure 4 The results of proliferation test and differentiation test of human subcutaneous preadipocytes when Compounds 7, 8 and 9 were used alone or in combination are shown.

[0032] Figure 5 The separation scheme for further purifying DEFr.3 and DEFr.5-DEFr.8 and isolating new compounds 12-19 is shown.

[0033] Figure 6 The figures show the results of proliferation assay and differentiation assay of compounds 11 (Cmpd11) to 19 (Cmpd19) on human subcutaneous preadipocytes. DETAILED DESCRIPTION

[0034] Next, various embodiments of the present invention will be described with reference to the accompanying drawings. It should be noted that the various embodiments described below do not limit the invention involved in the claims, and all of the various elements and their combinations described in the various embodiments are not necessarily required for the solution of the present invention.

[0035] (definition)

[0036] In this specification, "preadipocyte" refers to a cell derived from adipose tissue that has the ability to differentiate into adipocytes. Unlike mature adipocytes, preadipocytes do not contain fat in the cells yet, and are proliferative fibroblast-like cells. Mature adipocytes refer to cells that are differentiated from preadipocytes and are capable of accumulating fat in the cells, and their morphology is a state of accumulating fat in the cells. One of the causes of skin aging is the decline and reduction of subcutaneous fat caused by age and ultraviolet rays. If the volume of the subcutaneous fat layer decreases, the tension of the skin disappears, and unevenness is generated on the skin surface, leading to wrinkles and depressions. Therefore, mainly by increasing the preadipocytes of organisms such as humans and differentiating them into adipocytes, there is an effect of preventing, treating and / or improving the volume reduction of local skin (chest, lips, tear bags, back of hands, etc.). Moreover, it is believed that by increasing subcutaneous fat cells, the transparency and luster of the skin, together with nasolabial folds, etc., will also be improved.

[0037] The "proliferation and / or differentiation promoter of preadipocytes" in the present embodiment (hereinafter, sometimes referred to as "the preparation of the present embodiment") refers to a composition containing active ingredients for these uses, and is a raw material that can be added to cosmetics and pharmaceuticals. The preparation can be not only a liquid, but also a solid, and as a specific use, it can be not only a skin topical agent, but also an oral composition (for example, it can be made into a solid or a liquid).

[0038] (Active ingredient)

[0039] The agent for promoting proliferation and / or differentiation of preadipocytes according to the present embodiment contains a compound represented by the following formula (I) as an active ingredient.

[0040] [Chemistry 2]

[0041]

[0042] (In the formula, R is a hydrogen atom or an acyl group having 1 to 5 carbon atoms.) Here, the acyl group in the "acyl group having 1 to 5 carbon atoms" refers to a substituted carbonyl group, which is a straight-chain, branched, cyclic, saturated, or unsaturated aliphatic hydrocarbon group. Specifically, acetyl, propionyl, isopropionyl, isobutyryl, tigloyl, isovaleryl, pivaloyl, methacryloyl, acryloyl, crotonyl, and 2-methylcrotonyl can be cited. In a preferred embodiment, R in the above formula (I) is a hydrogen atom or an acyl group having 1 to 4 carbon atoms. As a further preferred embodiment, R is a hydrogen atom, acetyl, methacryloyl, or isobutyryl.

[0043] In one embodiment, the active ingredient of the present invention is 6-O-methacryloyl helianthus lactone and / or 6-O-isobutyryl helianthus lactone. Here, 6-O-methacryloyl helianthus lactone refers to compound 8 in which R represents a methacryloyl group in the compound represented by the above general formula (I). In addition, 6-O-isobutyryl helianthus lactone is compound 9 in which R represents an isobutyryl group in the compound represented by the above general formula (I).

[0044] In another embodiment, the active ingredient of the present invention is helianthus lactone or 6-O-acetyl helianthus lactone. Here, helianthus lactone refers to compound 12 in which R represents a hydrogen atom in the compound represented by the above general formula (I). In addition, 6-O-acetyl helianthus lactone is compound 13 in which R represents an acetyl group in the compound represented by the above general formula (I).

[0045] These compounds can be directly extracted from Arnica montana flowers, or can be synthesized by chemical modification of helianthus lactone extracted from medicinal plants such as Arnica montana as the parent compound. For example, a method of acetylation of the 6-hydroxyl group of helianthus lactone using acetic anhydride has been reported (see Maria F Beer et al., Molecules. 2019; 24(6): 1113, Scheme 2). Based on this, those skilled in the art can easily use helianthus lactone and acid anhydride to synthesize the above-mentioned compounds 8 and 9.

[0046] In the case of extracting the active ingredient of the present embodiment from the flowers of Arnica, "Arnica" is a plant of the Asteraceae family, the genus Arnica (scientific name Arnica montana). When manufacturing the extract of Arnica, as materials, for example, roots, rhizomes, leaves, stems, flowers, fruits, pericarps, seeds, whole herbs or mixtures thereof can be used. The extract of Arnica is prepared, for example, by extracting the material using a solvent after crushing the material fresh or dried. For example, 50 g of dried Arnica flowers are immersed in 1 kg of 30% 1,3-butanediol solution and extracted for 5 to 10 days in an environment of about 10°C to about 30°C. Alternatively, 50 g of dried Arnica flowers can be immersed in 1 kg of warm water at about 30°C to about 50°C and extracted for 2 to 10 hours. The solution obtained by the immersion is filtered using a predetermined filter material (Glass Fiber File Retention Time (Gf-75 manufactured by ADVANTEC) and Mixed Cellurose Ester (A045A047A manufactured by ADVANTEC) etc.). The filtered solution is allowed to stand at about 0°C to about 10°C for 5 to 10 days. The solution obtained by filtering again using a predetermined filter material is used as an extract of Arnica montana.

[0047] (Separation and purification of active ingredients)

[0048] The active ingredients of this embodiment are obtained by separation and extraction from Arnica montana extract using a solvent, separation using chromatography using various separation modes (ion exchange, hydrophilic adsorption, hydrophobic adsorption, size exclusion, ligand exchange, affinity, etc.), molecular weight separation and filtration using filter paper, membrane filter, ultrafiltration membrane, etc., pressurization or decompression, heating or cooling, drying, pH adjustment, deodorization, decolorization, long-term static storage, etc., and these can also be arbitrarily selected and combined. As an embodiment, it is preferred to combine column chromatography and preparative HPLC used in the examples described below. The structure of the purified compound can be determined by NMR and mass spectrometry.

[0049] (Proliferation and / or differentiation promoter of preadipocytes)

[0050] The content ratio of each active ingredient in the preparation of the present embodiment is not particularly limited. For example, the preparation of the present embodiment may contain only one of 6-O-methacryloyl helianthus lactone and 6-O-isobutyryl helianthus lactone, or may contain both of them in an arbitrary ratio. In a preferred embodiment, both 6-O-methacryloyl helianthus lactone and 6-O-isobutyryl helianthus lactone are contained, and more preferably they are contained in a ratio of approximately 1:1. The preparation of the present embodiment may also contain any compound in addition to these active ingredients. For example, the Asteraceae plants represented by Arnica montana contain a variety of helianthus lactones, their derivatives or their analogs. They are collectively referred to as lipophilic 15-carbon terpenoid compounds having a lactone ring, namely sesquiterpene lactones.

[0051] In folk remedies, sesquiterpene lactones are used as Chinese medicine for various symptoms. Its diversity is consistent with the diversity of its biological activity, indicating that it exerts anti-inflammatory, cytotoxic, anticancer, antibacterial, antifungal, insecticidal, and antiprotozoal effects. Therefore, in the case where the preparation of the present embodiment contains sesquiterpene lactones as other ingredients, it is necessary to pay attention to their various physiological activities, especially cytotoxicity. For example, as shown in the examples described later, arnica extract contains a variety of sesquiterpene lactones, and it is preferred to reduce the ratio of the components with strong cytotoxicity in them. As a cytotoxic helianthus lactone derivative, for example, 6-O-tiglyl helianthus lactone can be cited. On the other hand, by including a small amount of 6-O-tiglyl helianthus lactone than the active ingredient of the present embodiment, the differentiation of the above-mentioned adipocytes is sometimes promoted. Therefore, the preparation of the present embodiment preferably does not contain 6-O-tiglyl helianthus lactone or, in terms of mass ratio, contains 6-O-tiglyl helianthus lactone below the total amount of the active ingredient. This is because the cytotoxic effect caused by other components can be suppressed, and a synergistic effect with the active ingredient of the present embodiment can also be expected.

[0052] (Content of active ingredient contained in skin external preparation)

[0053] The preparation of this embodiment can be used as a skin external preparation as one of its uses. The content of each active ingredient in the skin external preparation is not particularly limited as long as it is an amount that can promote the proliferation and / or differentiation of preadipocytes, taking into account its administration mode and administration method, etc. For example, the content of each active ingredient is preferably 0.1×10 -9 mass % or more, more preferably 0.1×10 -7 mass % (about 0.1 ng / mL when used as an aqueous solution) or more, more preferably 0.3×10 -7 mass % or more, more preferably 1×10 -7 In addition, considering the cytotoxicity of the helical lactone derivative, it is preferably 1×10 -2 mass % or less, more preferably 1×10 -3 mass % or less, more preferably 1×10 -4 In one embodiment, when 6-O-tiglylhexanolide (Compound 7) is included, it is preferably about 1×10 -7 6-O-tiglylhexanolide is contained or not contained at a concentration of mass % or less.

[0054] (Forms of external skin preparations)

[0055] The skin external preparation of the present invention can be exemplified by 1) pharmaceuticals, 2) quasi-pharmaceuticals, 3) topical or systemic skin external preparations (e.g., lotions, emulsions, creams, ointments, lotions, oils, basic cosmetics such as facial masks, solid soaps, liquid soaps, face washes such as hand soaps, skin cleansers, massage agents, cleansing agents, hair removers, depilatory agents, shaving preparations, aftershaves, preshaves, shaving creams, foundations, lipsticks, blushers, eye shadows, eyeliners, mascara, etc.) in a form suitable for use such as ampoules, capsules, powders, granules, liquids, gels, bubbles, emulsions, sheets, mists, sprays, etc. 1) cosmetics, perfumes, nail polish, nail polish removers, poultices, plasters, tapes, sheets, patches, aerosols, etc.), 2) medicinal and / or cosmetic preparations for scalp and hair (e.g. shampoos, conditioners, hair treatments, hair pretreatments, permanent perm solutions, hair dyes, hair styling products, hair treatments, hair growth and nourishment products, poultices, plasters, tapes, sheets, aerosols, etc.), 3) other anti-armpit odor agents or deodorants, antiperspirants, sanitary products, sanitary napkins, wet wipes, etc.), 4) medicinal and / or cosmetic preparations for scalp and hair (e.g. shampoos, conditioners, hair treatments, hair pretreatments, permanent perm solutions, hair dyes, hair styling products, hair treatments, hair growth and nourishment products, poultices, plasters, tapes, sheets, aerosols, etc.), 5) bath preparations for use in bath water, 6) other anti-armpit odor agents or deodorants, antiperspirants, sanitary products, sanitary napkins, wet wipes, etc.

[0056] (Components of external skin preparations)

[0057] In such a preparation, the following components and additives can be arbitrarily selected and combined as needed within a range that does not impair the effects of the present invention. The amounts thereof in the formulation are not particularly limited, but are generally considered to be preferably about 0.0001 to 50%.

[0058] (1) Various oils and fats

[0059] Avocado oil, almond oil, fennel oil, perilla oil, olive oil, orange oil, Atlantic snapper oil (オレンジラファー oil), sesame oil, cocoa butter, chamomile oil, carrot oil, cucumber oil, beef tallow fatty acid, kusu nut oil, safflower oil, shea butter, liquid shea butter, soybean oil, camellia oil, corn oil, rapeseed oil, peach kernel oil, castor oil, cottonseed oil, peanut oil, turtle oil, mink oil, egg yolk oil, palm oil, palm kernel oil, wood wax, coconut oil, beef tallow, lard, squalene, squalane, batylane or hydrogenated products of these oils (hardened oils, etc.).

[0060] (2) Wax

[0061] Beeswax, carnauba wax, spermaceti, lanolin, liquid lanolin, reduced lanolin, hard lanolin, candelilla wax, montan wax, shellac wax, rice bran wax, etc.

[0062] (3) Mineral oil

[0063] Liquid paraffin, vaseline, paraffin, ozokerite, ceresin, microcrystalline wax, etc.

[0064] (4) Fatty acids

[0065] Natural fatty acids such as lauric acid, myristic acid, palmitic acid, stearic acid, behenic acid, oleic acid, linoleic acid, linolenic acid, docosahexaenoic acid, eicosapentaenoic acid, 12-hydroxystearic acid, undecylenic acid, tall oil, lanolin fatty acid, and synthetic fatty acids such as isononanoic acid, caproic acid, 2-ethylbutyric acid, isovaleric acid, 2-methylvaleric acid, 2-ethylhexanoic acid, and isovaleric acid.

[0066] (5) Alcohols

[0067] Natural alcohols such as ethanol, isopropyl alcohol, lauryl alcohol, cetyl alcohol, stearyl alcohol, oleyl alcohol, lanolin alcohol, cholesterol, phytosterols, phenoxyethanol, and synthetic alcohols such as 2-hexyldecanol, isostearyl alcohol, and 2-octyldodecanol.

[0068] (6) Polyols

[0069] Ethylene oxide, ethylene glycol, diethylene glycol, triethylene glycol, ethylene glycol monoethyl ether, ethylene glycol monobutyl ether, diethylene glycol monomethyl ether, diethylene glycol monoethyl ether, polyethylene glycol, propylene oxide, propylene glycol, polypropylene glycol, 1,3-butylene glycol, pentylene glycol, glycerol, pentaerythritol, threitol, arabitol, xylitol, ribitol, galactitol, sorbitol, mannitol, lactitol, maltitol, etc.

[0070] (7) Esters

[0071] Isopropyl myristate, isopropyl palmitate, butyl stearate, hexyl laurate, myristyl myristate, oleyl oleate, decyl oleate, octyldodecyl myristate, hexyl decyl dimethyl octanoate, cetyl lactate, myristyl lactate, diethyl phthalate, dibutyl phthalate, lanolin acetate, ethylene glycol monostearate, propylene glycol monostearate, propylene glycol dioleate, etc.

[0072] (8) Metal soaps

[0073] Aluminum stearate, magnesium stearate, zinc stearate, calcium stearate, zinc palmitate, magnesium myristate, zinc laurate, zinc undecylenate, etc.

[0074] (9) Gum, sugar or water-soluble polymer compounds

[0075] Arabic gum, benzoin gum, dammar gum, guaiac, Irish moss, karaya gum, tragacanth gum, carob bean gum, quince seed, agar, casein, lactose, fructose, sucrose or its esters, trehalose or its derivatives, dextrin, gelatin, pectin, starch, carrageenan, carboxymethyl chitin or chitosan, hydroxyalkyl (C2-C4) chitin or chitosan to which alkylene (C2-C4) oxides such as ethylene oxide are added, low molecular weight chitin or chitosan, chitosan salts, chitosan sulfate or chitosan, phosphorylated chitosan cellulose or chitosan, alginic acid or its salt, hyaluronic acid or its salt, chondroitin sulfate or its salt, heparin, ethyl cellulose, methyl cellulose, carboxymethyl cellulose, carboxyethyl cellulose, sodium carboxyethyl cellulose, hydroxyethyl cellulose, hydroxypropyl cellulose, nitrocellulose, crystalline cellulose, polyvinyl alcohol, polyvinyl methyl ether, polyvinyl pyrrolidone, polyvinyl methacrylate, polyacrylate, polyalkylene oxide such as polyethylene oxide or polypropylene oxide or their cross-linked polymers, carboxyvinyl polymers, polyethylene imine, etc.

[0076] (10) Surfactants

[0077] Anionic surfactants (alkyl carboxylates, alkyl sulfonates, alkyl sulfate ester salts, alkyl phosphate ester salts), cationic surfactants (alkyl amine salts, alkyl quaternary ammonium salts), amphoteric surfactants: carboxylic acid type amphoteric surfactants (amino type, betaine type), sulfate type amphoteric surfactants, sulfonic acid type amphoteric surfactants, phosphate type amphoteric surfactants, nonionic surfactants (ether type nonionic surfactants, ether ester type nonionic surfactants, ester type nonionic surfactants, block polymer type nonionic surfactants, nitrogen-containing nonionic surfactants), other surfactants (natural surfactants, protein hydrolyzate derivatives, polymer surfactants, titanium / silicon-containing surfactants, fluorinated carbon surfactants), etc.

[0078] (11) Various vitamins

[0079] Vitamin A family: retinol, retinal (vitamin A1), dehydroretinal (vitamin A2), carotene, lycopene (provitamin A), vitamin B family: thiamine hydrochloride, thiamine sulfate (vitamin B1), riboflavin (vitamin B2), pyridoxine (vitamin B6), cyanocobalamin (vitamin B12), folic acid, niacin, pantothenic acid, biotin, choline, inositol, vitamin C family: vitamin C acid or its derivatives, vitamin D family: ergocalciferol (vitamin D2), cholecalciferol Calciferol (vitamin D3), dihydrotachysterol, vitamin E family: vitamin E or its derivatives, ubiquinones, vitamin K family: phytomenadione (vitamin K1), menaquinone (vitamin K2), 2-menadione (vitamin K3), hydromenadione (vitamin K4), other essential fatty acids (vitamin F), carnitine, ferulic acid, γ-oryzanol, orotic acid, vitamin P (rutin, erythritol, hesperidin), vitamin U, etc.

[0080] (12) Various amino acids

[0081] Valine, leucine, isoleucine, threonine, methionine, phenylalanine, tryptophan, lysine, glycine, alanine, asparagine, glutamine, serine, cysteine, cystine, tyrosine, proline, hydroxyproline, aspartic acid, glutamic acid, hydroxylysine, arginine, ornithine, histidine, and their sulfates, phosphates, nitrates, citrates, or amino acid derivatives such as pyrrolidonecarboxylic acid, etc.

[0082] (13) Various additives derived from plant or animal raw materials

[0083] They can be processed by conventional methods according to the type and form of the product to be added (for example, arbitrarily selecting and combining crushing, pulverizing, washing, hydrolysis, fermentation, refining, pressing, extraction, fractionation, filtration, drying, powdering, granulation, dissolution, sterilization, pH adjustment, deodorization, decolorization, etc.), and can be arbitrarily selected and supplied from various raw materials.

[0084] The present invention is further described in detail with reference to the following examples, but the present invention is not limited by these examples. It should be noted that in the following examples, the unit % representing the amount of each component added refers to mass %.

[0085] Example

[0086] [Example 1]

[0087] (Isolated components of Arnica montana hot water extract)

[0088] Dried Arnica flowers are immersed in warm water at about 50°C for 5 hours. The solution obtained by the immersion is filtered using a specified filter material (glass filter paper, Gf-75, manufactured by ADVANTEC) and a cellulose mixed ester membrane filter, A045A047A, manufactured by ADVANTEC). The filtered solution is concentrated using an evaporator, and then made into powder using a freeze dryer and used as an Arnica extract. Figure 1 The separation scheme shown separates the components of the Arnica montana extract.

[0089] The Arnica montana hot water extract (211.8 g) was suspended in distilled water (500 mL), and liquid-liquid distribution was performed using diethyl ether (5×250 mL, S01), ethyl acetate (5×250 mL, S02), and butanol (5×250 mL, S03), respectively, to obtain a diethyl ether fraction (DEFr. 3.9 g), an ethyl acetate fraction (EFr. 2.8 g), a butanol fraction (BFr. 10.8 g) and a water fraction (WFr. The yield could not be determined).

[0090] The most active DEFr. (3.9 g) was subjected to silica gel open column chromatography (57 mm φ × 355 mm L (diameter 57 mm, length 355 mm), S04), and eluted with hexane: acetone = 3: 1, 3: 2, 1: 1. The eluate was analyzed by TLC, and 13 fractions (DEFr. 1 to DEFr. 13) were obtained. DEFr. 9 (281.9 mg) was subjected to preparative HPLC (condition 1, S06), and compound 1 (6.4 mg, retention time 19.7 min) was obtained. Highly active DEFr. 4 (659.2 mg) was observed in the fine fractions and was subjected to LH-20 gel open column chromatography (20 mm φ × 515 mm L, S05), and eluted with chloroform: methanol = 1: 1. The eluate was analyzed by TLC, and 7 fractions (DEFr. 4-1 to DEFr. 4-7) were obtained.

[0091] DEFr.4-2 (462.6 mg) was subjected to silica gel open column chromatography (35 mmφ×363 mmL, S07) and eluted with hexane:chloroform:ethyl acetate = 1:1:0.1, 1:1:0.3, 1:1:1, and chloroform:methanol = 1:1. The eluate was analyzed by TLC to obtain 13 fractions (DEFr.4-2-1 to DEFr.4-2-13).

[0092] DEFr.4-2-6 (66.5 mg) was subjected to preparative HPLC (condition 2, S09) to obtain compound 2 (5.1 mg, retention time 31.2 min), compound 3 (13.5 mg, retention time 31.8 min), and compound 4 (19.3 mg, retention time 32.0 min).

[0093] DEFr.4-2-2 (348.4 mg) was subjected to ODS gel open column chromatography (30 mmφ×112 mmL, S08) and eluted with water:methanol=1:1, 1:2, and 0:1. The eluate was analyzed by TLC to obtain 13 fractions (DEFr.4-2-2-1 to DEFr.4-2-2-13).

[0094] DEFr.4-2-2-4 (50.9 mg) was subjected to preparative HPLC (Condition 3, S10) to obtain Compound 5 (5.1 mg, retention time 30.8 min) and Compound 6 (10 mg, retention time 31.6 min).

[0095] DEFr.4-2-2-8 (77.2 mg) was subjected to preparative HPLC (Condition 4, S12) to obtain Compound 7 (43.2 mg, retention time 33.3 min).

[0096] DEFr.4-2-2-6, 7 (80.9 mg) were subjected to preparative HPLC (condition 5, S11) to give compound 8 (9.5 mg, retention time 31.6 min), compound 9 (10.9 mg, retention time 32.2 min), and compound 10 (15.9 mg, retention time 32.5 min).

[0097] (Structural analysis of isolated compounds)

[0098] Structural analysis of compound 1

[0099] DEFr.9-1 (6.4 mg) was dissolved in deuterated methanol and subjected to FT-NMR analysis. It was also dissolved in methanol and subjected to MALDI-TOF-MS analysis.

[0100] Structural analysis of compound 2

[0101] DEFr.4-2-6-1 (5.1 mg) was dissolved in deuterated acetone and deuterated chloroform and subjected to FT-NMR analysis, or dissolved in methanol and subjected to MALDI-TOF-MS analysis.

[0102] Structural analysis of compound 3

[0103] DEFr.4-2-6-2 (13.5 mg) was dissolved in deuterated acetone and deuterated chloroform and subjected to FT-NMR analysis, or dissolved in methanol and subjected to MALDI-TOF-MS analysis.

[0104] Structural analysis of compound 4

[0105] DEFr.4-2-6-3 (19.1 mg) was dissolved in deuterated acetone and deuterated chloroform and subjected to FT-NMR analysis, or dissolved in methanol and subjected to MALDI-TOF-MS analysis.

[0106] Structural analysis of compound 5

[0107] DEFr.4-2-2-4-1 (5.1 mg) was dissolved in deuterated chloroform and subjected to FT-NMR analysis, or dissolved in methanol and subjected to MALDI-TOF-MS analysis.

[0108] Structural analysis of compound 6

[0109] DEFr.4-2-2-4-2 (10.8 mg) was dissolved in deuterated chloroform and subjected to FT-NMR analysis, or dissolved in methanol and subjected to MALDI-TOF-MS analysis.

[0110] Structural analysis of compound 7

[0111] DEFr.4-2-2-8-1 (43.2 mg) was dissolved in deuterated chloroform and subjected to FT-NMR analysis, or dissolved in methanol and subjected to MALDI-TOF-MS analysis.

[0112] Structural analysis of compound 8

[0113] DEFr.4-2-2-6-1 (9.5 mg) was dissolved in deuterated chloroform and subjected to FT-NMR analysis, or dissolved in methanol and subjected to MALDI-TOF-MS analysis.

[0114] Structural analysis of compound 9

[0115] DEFr.4-2-2-6-2 (10.9 mg) was dissolved in deuterated chloroform and subjected to FT-NMR analysis, or dissolved in methanol and subjected to MALDI-TOF-MS analysis.

[0116] Structural analysis of compound 10

[0117] DEFr.4-2-2-6-3 (3.2 mg) was dissolved in deuterated chloroform and subjected to FT-NMR analysis, or dissolved in methanol and subjected to MALDI-TOF-MS analysis.

[0118] The purity of the target compound in the sample used for the above structural analysis was analyzed by high performance liquid chromatography (HPLC) described later, and the results are shown in Table 1 below.

[0119] [Table 1]

[0120] Sample name Target compound Purity of the target compound (%) DEFr.9-1(Cmpd 1) Compound 1 97.35％ DEFr.4-2-6-1 - 99.55％ DEFr.4-2-6-2(Cmpd 3) Compound 3 99.52％ DEFr.4-2-6-3 (Cmpd 4) Compound 4 99.95％ DEFr.4-2-2-4-1(Cmpd 5) Compound 5 100％ DEFr.4-2-2-4-2 (Cmpd 6) Compound 6 100％ DEFr.4-2-2-8-1(Cmpd 7) Compound 7 95.05％ DEFr.4-2-2-6-1(Cmpd 8) Compound 8 100％ DEFr.4-2-2-6-2 (Cmpd 9) Compound 9 92.74％ DEFr.4-2-2-6-3(Cmpd 10) Compound 10 66.26％

[0121] <High Performance Liquid Chromatography (HPLC)>

[0122] Installation: SHIMADZU

[0123] Pump: LC-20AD

[0124] Diode array detector: SPD-M20A

[0125] Column oven: CTO-20AD

[0126] System controller: CBM-20A

[0127] Degasser: DGU-20A

[0128] Automatic sample injector: SIL-20A

[0129] Column: Inertsil (registered trademark) ODS-3, 5 (4.6 mmφ×250 mmL) Injection volume: 10 μl

[0130] Analysis time: 60min

[0131] Detector wavelength: 210~600nm

[0132] Flow rate: 1.0ml / min

[0133] Gradient program: methanol: 0.05% TFAaq. = 5%: 95% → 100%: 0% (40 min)

[0134] <Preparative HPLC>

[0135] Installation: JASCO

[0136] Pump: 880-PU

[0137] Detector: 870-UV

[0138] Degasser: 880-51

[0139] System controller: 880-30

[0140] Column: Inertsil (registered trademark) ODS-3, 5 (20 mmφ×250 mmL) Injection volume: 350 μl

[0141] Detector wavelength: 280.0nm

[0142] Flow rate: 9.0ml / min

[0143] (Condition 1)

[0144] Gradient program: methanol: 0.05% TFA aq. = 25:75 → 100:0 (30 min)

[0145] (Condition 2)

[0146] Gradient program: methanol: 0.05% TFA aq. = 60:40 → 85:15 (30 min)

[0147] (Condition 3)

[0148] Gradient program: methanol: water = 60:40 (30 min)

[0149] (Condition 4)

[0150] Gradient program: acetonitrile: water = 60:40 → 85:15 (30 min)

[0151] (Condition 5)

[0152] Gradient program: acetonitrile: 0.05% TFA aq. = 50:50 (30 min)

[0153] (Condition 6)

[0154] Gradient program: methanol: water = 65:35 (30 min)

[0155] <FT-NMR>

[0156] Device: JEOL EC 600MHz (JEOL, TOKYO, JAPAN)

[0157] Solvent: Chloroform-d (Cambridgr Isotope Laboratories, Inc. CAS 865-49-6)

[0158] Methanol-d4 (Kanto Chemical Co., Ltd., CAS 25221-96)

[0159] Acetone-d6 (SIGMA-ALDRICH, CAS666-52-4)

[0160] <Matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF-MS)>

[0161] Installation: SHIMADZU BIOTECH AXIMA RESONANCE

[0162] Ion mode: positive ion mode

[0163] Quality range: Low100+, Low300+

[0164] Power:120~150

[0165] Matrix: 2,5-dibenzoic acid (DHB)

[0166] <Polarimeter>

[0167] Device: JASCO-P2300 system

[0168] Solvent: methanol, chloroform

[0169] For each sample, the 13 C-NMR spectroscopy, 1 The structures of Compound 1 and Compounds 3 to 10 estimated from the H-NMR spectrum, COSY spectrum, HMBC spectrum, HMQC spectrum and MALDI-TOF-MS spectrum are shown below.

[0170] [Chemistry 3]

[0171]

[0172] (Human subcutaneous preadipocyte proliferation assay)

[0173] By adding the above-mentioned purified fractions to human subcutaneous preadipocytes, it is confirmed whether cell proliferation is promoted. Each purified fraction used in this evaluation is prepared as described in Tables 2 and 3 based on the mass recovered from the arnica flower hot water extract in the proportion existing in the original extract. First, each purified fraction is dissolved in DMSO to make a stock solution. After dissolving it in 30% BG in a manner of 0.75%, it is diluted 1000 times or 3000 times and added to the cells. After dissolving 50 mg of the powder of the arnica flower hot water extract as the mother in 500 μL of 30% BG, it is diluted in 30% BG in a manner of 0.75%, and DMSO is further added in a manner of 0.75%, and the solution obtained is diluted 1000 times or 3000 times and added to the cells. In addition, as a comparison object, DMSO is added in a manner of 0.75% in 30% BG and prepared, diluted 1000 times or 3000 times and added to the cells.

[0174] [Table 2]

[0175]

[0176] [Table 3]

[0177]

[0178] Human subcutaneous preadipocytes (manufactured by Lonza, PT-5020) were cultured at 1×10 3 (cells) / well were seeded in a 96-well plate and cultured in a 5% CO2 incubator at 37°C for 16 hours. Then, each purified fraction was added in such a manner as to become the above concentration, and cultured in a 5% CO2 incubator at 37°C for 72 hours. After culture, the number of cells in the well was measured. As a control for this experiment, a well was set to add 30% 1,3-butanediol, an extraction solvent that does not contain each purified fraction, and the number of cells in the set well was also measured. The number of cells in the set well was set to 100, and the ratio (relative value) of the number of cells in the well to which each purified fraction was added was calculated.

[0179] (Differentiation test of human subcutaneous preadipocytes)

[0180] It is known that fat droplets are stored in cells by differentiation from preadipocytes to adipocytes (Obesity Research vol. 13, No. 1, 2007, pp. 84-86). By adding each of the above-mentioned purified fractions to human subcutaneous preadipocytes, it was confirmed whether differentiation into adipocytes was promoted by the formation of fat droplets. Each purified fraction used in this evaluation was prepared based on the mass recovered from the Arnica montana hot water extract in a manner that would be present in the original extract, and was diluted 1000-fold or 3000-fold and added to the cells.

[0181] Human subcutaneous preadipocytes (manufactured by Lonza, PT-5020) were cultured at 1×10 4 (cells) / well were seeded in a 96-well plate and cultured in a 5% CO2 incubator at 37°C for 16 hours. After visually confirming that the cells had become confluent under a microscope, a differentiation induction medium (Lonza, PT-8002) was added at a concentration 1 / 2 times that recommended by Lonza. At this time, each purified fraction was added so as to achieve the above concentration, and the cells were cultured in a 5% CO2 incubator at 37°C for 10 days. After visually confirming the accumulation of fat droplets under a microscope, the culture medium was removed and the wells were washed twice with PBS. After the cells were fixed with 10% neutral buffered formalin (Wako Pure Chemical Industries, Ltd., 062-01661), the fat droplets were stained with Oil Red O (Wako Pure Chemical Industries, Ltd., 154-02072) saturated with 60% isopropanol. After washing the wells with purified water, the stained fat droplets were extracted with 100% isopropanol and the absorbance at 510 nm was measured. The absorbance value of the wells to which 30% 1,3-butanediol as an extraction solvent was added in place of each purified fraction to give the above concentration was set as 1, and the ratio (relative value) of the absorbance of the wells to which the Arnica extract was added was calculated as the differentiation ratio (relative value).

[0182] The results are shown in Figures 2 to 4 .exist Figures 2 to 4 In the formula, “Parent” refers to the Arnica montana hot water extract used as a purification raw material, and “Cont” refers to 30% 1,3-butylene glycol. Figure 2As a result, the activity of promoting the proliferation and differentiation of human subcutaneous preadipocytes was found in the fraction DEFr. obtained by diethyl ether extraction (S01) of the Arnica montana extract and the fraction DEFr.4 obtained by silica gel open column chromatography separation (S04). Therefore, the DEFr.4 fraction was further separated by silica gel open column chromatography and preparative HPLC. The results of the above-mentioned determination of the obtained fractions (DEFr.2, Cmpd1, Cmpd3, Cmpd4, Cmpd5, Cmpd6, Cmpd7, Cmpd8, Cmpd9 and Cmpd10 described in Table 3) are shown in FIG. Figure 3 .Depend on Figure 3 The results showed that Cmpd7 (including compound 7), Cmpd8 (including compound 8) and Cmpd9 (including compound 9) have the activity of promoting the proliferation and differentiation of human subcutaneous preadipocytes. It is believed that the reason why the proliferation activity of human subcutaneous preadipocytes is reduced when Cmpd7 is diluted 1000 times is due to the cytotoxicity of the compound.

[0183] Therefore, the results of the proliferation test and differentiation test of human subcutaneous preadipocytes when Cmpd7 (containing compound 7), Cmpd8 (containing compound 8) and Cmpd9 (containing compound 9) were used alone or in combination are shown in Figure 4 It should be noted that Figure 4 In the formula, "Cmpd7+8" means adding Cmpd7 and Cmpd8 at the same time, "Cmpd7+9" means adding Cmpd7 and Cmpd9 at the same time, "Cmpd8+9" means adding Cmpd8 and Cmpd9 at the same time, and "Cmp7+8+9" means adding Cmpd7, Comp8 and Cmpd9 at the same time. Figure 4 The results showed that the combination of Cmpd8 and Cmpd9 showed the strongest activity in promoting the proliferation and differentiation of human subcutaneous preadipocytes. Figures 2 to 4 In the table, * indicates a significant difference of p<0.05 by Dunnett's test (one-sided only) relative to Cont, and ** indicates a significant difference of p<0.01 by Dunnett's test (one-sided only) relative to the same Cont.

[0184] [Example 2]

[0185] From the 13 fractions (DEFr.1 to DEFr.13) obtained by separating the diethyl ether soluble portion obtained in Example 1 by silica gel open column chromatography, each fraction of DEFr.3 and DEFr.5 to DEFr.8 was further purified to separate new compounds 12 to 19. The separation scheme for separating these new compounds is shown in Figure 5 .

[0186] DEFr.3 (340.0 mg) was subjected to ODS gel open column chromatography (30 mmφ×112 mmL, S14) and eluted with water:methanol=3:2. DEFr.3-8 (156.0 mg) in which activity was observed in the fine fraction was subjected to preparative HPLC (acetonitrile:water=39:61, S15) to obtain compound 17 (8.3 mg). On the other hand, the obtained fraction DEFr.3-8-5 was further subjected to preparative HPLC (hexane:2-propanol=90:10, S16) to obtain compound 16 (15.8 mg).

[0187] DEFr.5 (504.6 mg) was subjected to LH-20 gel open column chromatography (20 mmφ×515 mmL, S17) and eluted with methanol:chloroform=3:2. The eluate was analyzed by TLC to obtain two fractions (DEFr.5-2 and DEFr.5-3). The fraction DEFr.5-2 (167.0 mg) was subjected to preparative HPLC (methanol:water=45:55, S18) to obtain compound 13 (11.2 mg). On the other hand, the obtained fraction DEFr.5-2-1 was further subjected to preparative HPLC (acetonitrile:water=30:70, S19) to obtain compound 14 (2.1 mg). Fraction DEFr.5-3 (130.1 mg) was subjected to ODS gel open column chromatography (S20). Water:methanol=2:3, 1:1 and 1:0 were used for elution, respectively. The obtained fraction DEFr.5-3-23 (5.0 mg) was further subjected to preparative HPLC (methanol:water=40:60, S21) to obtain compound 15 (2.9 mg), and the fraction DEFr.5-3-4 (40.0 mg) was further subjected to preparative HPLC (methanol:water=25:75, S22) to obtain compound 15 (13.4 mg).

[0188] DEFr.6 (299.0 mg) was subjected to ODS gel open column chromatography (30 mmφ×112 mmL, S23) and eluted with water:methanol=1:1, 1:2, 0:1. The obtained fraction DEFr.6-3 (59.1 mg) was subjected to silica gel column chromatography and eluted with chloroform:methanol=20:1 (S24), and one eluted fraction DEFr.6-3-1 (40.0 mg) was further subjected to preparative HPLC (methanol:water=40:60, S25) to obtain compound 12 (9.5 mg).

[0189] DEFr.7 (515.0 mg) was subjected to LH-20 gel open column chromatography (20 mmφ×515 mmL, S05) and eluted with chloroform:methanol=1:1. The eluate was analyzed by TLC to obtain 6 fractions (DEFr.7-1 to DEFr.7-6). DEFr.7-4 (32.0 mg) was subjected to preparative HPLC and eluted with methanol:water (0.05% TFA)=30:70, S27) to obtain compound 18 (18.2 mg).

[0190] DEFr.8 (548.2 mg) was subjected to LH-20 gel open column chromatography (20 mmφ×515 mmL, S05) and eluted with chloroform:methanol=1:1. The obtained fraction DEFr.8-7 (25.3 mg) was subjected to preparative HPLC and eluted with methanol:water (0.05% TFA)=25:75, S29) to obtain compound 19 (11.3 mg).

[0191] (Structural analysis of isolated compounds)

[0192] By the same method as in Example 1, 13 C-NMR spectroscopy, 1 The structures of Compounds 11 to 19 estimated from the H-NMR spectrum, COSY spectrum, HMBC spectrum, HMQC spectrum, and MALDI-TOF-MS spectrum are shown below.

[0193] [Chemistry 4]

[0194]

[0195] The proliferation test and differentiation test of human subcutaneous preadipocytes were carried out in the same manner as in Example 1. The results are shown in Tables 4 and 5 below. Figure 6 In Table 4 and Figure 6 In the sample names, "KS0019" and "KS0020" respectively represent different batches of Arnica extracts. "BG / DMSO" represents a group to which 30% 1,3-butanediol and DMSO, which are extraction solvents not containing purified fractions, were added. Cmpd11 to Cmpd19 contain compounds 11 to 19, respectively. All samples were adjusted in concentration so that the concentration of the compound was the same as that in the Arnica extract, diluted 1000-fold, and added to the cells.

[0196] [Table 4]

[0197]

[0198] According to Table 4 and Figure 6The results showed that Cmpd12 (containing compound 12) and Cmpd13 (containing compound 13) significantly promoted the differentiation of human subcutaneous preadipocytes.

[0199] It should be noted that in Figure 6 In the figure, * indicates a significant difference of p < 0.05 by Dunnett's test (one-sided only) relative to BG / DMSO, ** indicates a significant difference of p < 0.01 by Dunnett's test (one-sided only) relative to the same BG / DMSO, *** indicates a significant difference of p < 0.001 by Dunnett's test (one-sided only) relative to the same BG / DMSO.

[0200] Industrial Applicability

[0201] The agent for promoting proliferation and / or differentiation of preadipocytes of the present invention can be industrially utilized as cosmetics, foods, pharmaceuticals, etc. and / or raw materials to be blended therewith.

Claims

1. Use of a compound represented by the following formula (I) in the manufacture of a skin topical preparation for promoting the proliferation and / or differentiation of preadipocytes, wherein the topical preparation contains an effective amount of a preadipocyte proliferation and / or differentiation promoter and does not contain 6-O-tiglyloyl-helidolide in an amount exceeding the total amount of the active ingredient by mass ratio, ; In formula (I), R is methacryloyl or isobutyryl, The agent for promoting proliferation and / or differentiation of preadipocytes is a preparation containing the compound represented by the above formula (I) as an active ingredient, wherein the compound is 6-O-methacryloyl helianthus lactone and 6-O-isobutyryl helianthus lactone.

2. The use according to claim 1, wherein The content of 6-O-methacryloyl helianthus lactone and 6-O-isobutyryl helianthus lactone is at least 0.1×10 -7 quality%.

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